The object of this proposal is to continue and extend our studies on brain peptidases and define their role in neuropeptide metabolism. Our recent studies have focused on the enzymology of thyrotropin-releasing hormone (TRH) metabolism. We have synthesized active-site directed inhibitors to prolyl endopeptidase and pyroglutamyl peptidase I, two TRH degrading enzymes. The use of these inhibitors has delineated a third, more specific TRH degrading enzyme. The latter enzyme termed pyroglutamyl peptidase Ii or "Thyroliberinase" is highly localized to brain where it is found on synaptosomal membranes. It exhibits an unusual degree of substrate specificity and may be the first true neuropeptide-specific peptidase to be characterized. We have purified this enzyme 5000 fold from rabbit brain and have developed a convenient assay to measure its activity. We propose to further characterize this enzyme. Data from structure- activity relationship studies and from computer assisted molecular modeling will be used to design active-site directed inhibitors to pyroglutamyl peptidase II. The enzyme will be localized in collaborative studies by immunohistochemistry and its localization compared to that of TRH and TRH receptors. Inhibitors of the three TRH metabolizing enzymes will be used to block TRH degradation in vivo in an attempt to measure TRH turnover, and in vitro to enhance the recovery of TRH released from brain slices. We have shown that pyroglutamyl peptidase I is regulated in GH3 cells. We will investigate whether the regulation of this enzyme affects prolactin secretion or synthesis in GH3 cells. Pyroglutamyl peptidase I will be purified to homogeneity, characterized, and antisera generated for immunohistochemical localization. Inhibitors of pyroglutamyl peptidase I will be administered to rats to determine if its inactivation can lead to an elevation of serum thyrotropin levels. Specific inhibitors of neuropeptide metabolizing enzymes are important tools for the study of neuropeptide function. Moreover, such compounds may possess valuable therapeutic properties. Inhibitors of TRH degrading enzymes could be used to potentiate the transient effects of TRH in the treatment of amyotrophic lateral sclerosis.